Pharmacokinetics of a drug candidate is a critical parameter and often largely determines whether or not the drug candidate will be further developed into a drug or used for a therapeutic application. In particular, pharmacokinetic studies of protein- and peptide-based therapeutics, including antibodies, have demonstrated that such therapeutics have varying serum half lives, and those peptides and proteins with a short serum half-life, although having a promising therapeutic potential, are thus often unsuitable for further drug development. For example, albumin and gamma immunoglobulins (IgGs) are known to have very long serum half lives of up to 20 days [1]. In addition, the Fc domain of other IgGs can be engineered to alter their binding interactions to neonatal Fc receptor as a method of prolonging serum half-life [2]. However, many other human proteins, such as insulin, antibody fragments such as antigen binding fragments (Fabs) or single chain variable fragment (scFvs), and short peptides usually have much shorter serum half-lives ranging from minutes to approximately only 1 hour. An efficient, cost effective and safe way of extending serum half lives of proteins and peptides with short half-lives is therefore critical for these molecules to become therapeutic drugs, diagnostic tools, etc.
Several strategies have been developed in an effort to prolong the serum half-life of proteins and other peptidic molecules that are short-lived in serum to avoid clearance of such therapeutic or diagnostic proteins from circulation. Several technologies employed to facilitate serum half-life extension of proteins and peptidic molecules include conjugation with a chemical attachment such as polyethylene glycol (i.e. pegylation) [3], fusion to the Fc region of an antibody [4], and fusion to a protein naturally having a long serum half life, such as albumin [5]. Unfortunately, these technologies suffer from complications including complex manufacturing and characterization processes, low expression levels, and undesired functions of the generated molecules.
Another approach that has been more recently developed to extend the serum half-life of proteins and other peptidic molecules employs the use of antibody fragments against serum proteins. Albumin, due mainly to its high serum concentration and long serum half-life, has been the most selected target for this purpose. An isolated domain antibody against human albumin was shown to prolong the serum half-life of interferon (IFN)-α2b after the two molecules were fused at the genetic level and expressed as a fusion protein [6, 7]. The serum half-life of the newly generated fusion protein molecule is not only longer than that of IFN-α2b, but even longer than that of the fusion protein of albumin and IFN-α2b.
Heavy chain variable domains of camelid heavy chain antibodies (HCAbs), known as VHH or single domain antibodies (sdAbs), have also been exploited for this purpose. For example, a sdAb against human albumin was shown to extend the serum half-life of an anti-TNFα sdAb fragment from less than one hour to over two days [8].
Another serum protein with a long half life is transferrin. Transferrin is a plasma glycoprotein that transfers iron ions and has a serum concentration of approximately 3 g/L and serum half-life of 7-8 days. Thus, transferrin is an ideal fusion partner to extend the serum half life of peptidic molecules with unsatisfactory pharmatokinetics. Studies have shown that fusion to transferrin significantly extended the serum half-life of both glucagon-like peptide 1 (GLP1) [9] and acetylcholine receptor [10].
However, the use of antibodies and antibody fragments such as sdAbs against transferrin, for increasing the serum half-life of peptidic molecules has not been reported. New methods for increasing the serum half-life of proteins and for producing proteins with improved serum half-life, that are efficient, cost-effective, and produce such proteins in high yield would facilitate the development of novel protein-based diagnostics or therapeutics. Embodiments of the present invention relate to such methods.